U.S. patent application number 13/057695 was filed with the patent office on 2011-09-15 for method and composition for controlling gene expression.
Invention is credited to Karl Deisseroth, Feng Zhang.
Application Number | 20110223635 13/057695 |
Document ID | / |
Family ID | 41669251 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110223635 |
Kind Code |
A1 |
Deisseroth; Karl ; et
al. |
September 15, 2011 |
Method and Composition for Controlling Gene Expression
Abstract
A composition for expressing a protein in cells is provided. In
certain embodiments, a circular expression vector provided herein
comprises: a promoter, a coding sequence encoding a protein of
interest, in which the coding sequence is in a reversed 3'-5'
orientation, a transcription termination sequence, and at least a
first recombination site and a second recombination site flanking
the coding sequence. A method for using the disclosed composition
and a kit comprising the composition are also provided herein.
Inventors: |
Deisseroth; Karl; (Palo
Alto, CA) ; Zhang; Feng; (Palo Alto, CA) |
Family ID: |
41669251 |
Appl. No.: |
13/057695 |
Filed: |
August 11, 2009 |
PCT Filed: |
August 11, 2009 |
PCT NO: |
PCT/US09/53474 |
371 Date: |
April 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61087903 |
Aug 11, 2008 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2840/203 20130101; C12N 2740/15043 20130101; C12N 2800/40
20130101; C12N 2750/14043 20130101; C12N 2750/14143 20130101; C12N
2800/30 20130101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325 |
International
Class: |
C12P 21/00 20060101
C12P021/00; C12N 15/85 20060101 C12N015/85; C12N 5/071 20100101
C12N005/071 |
Claims
1. A circular expression vector, comprising: a promoter; a coding
sequence encoding a protein of interest, wherein the coding
sequence is in a reversed 3'-5' orientation; a transcription
termination sequence; and at least a first recombination site and a
second recombination site flanking the coding sequence.
2. The vector of claim 1, wherein said coding sequence when
inverted by a recombinase is incapable of subsequent
recombination.
3. The vector of claim 1, wherein the first recombination site is
an attP site and the second recombination site is an attB site.
4. The vector of claim 3, wherein said attP site and said attB site
recombine in the presence of a unidirectional, site-specific
recombinase.
5. The vector of claim 4, wherein said unidirectional,
site-specific recombinase is phiC31 integrase.
6. The vector of claim 1, wherein the vector further comprises a
third recombination site interposed between said first
recombination site and said coding sequence and a fourth
recombination site interposed between said second recombination
site and said transcription termination region, wherein said first
recombination site and said second recombination site recombine in
the presence of a recombinase and said third recombination site and
said fourth recombination site recombine in the presence of a
recombinase.
7. The vector of claim 6, wherein said first recombination site and
second recombination site are LoxP sites and said third
recombination site and fourth recombination site are Lox2722
sites.
8. The vector of claim 6, wherein said recombinase is Cre
recombinase or Flp recombinase.
9. The vector of claim 1, wherein said circular expression vector
is an episomal vector.
10. The vector of claim 7, wherein said episomal vector is an
adeno-associated vector.
11. The vector of claim 7, wherein said episomal vector is an
EBNA-1 based episomal vector.
12. A method of expressing a protein of interest in a cell,
comprising: transfecting a cell with an episomal circular
expression vector, comprising: a promoter; a coding sequence
encoding a protein of interest, wherein the coding sequence is in a
reversed 3'-5' orientation; a transcription termination sequence;
and at least a first recombination site and a second recombination
site flanking the coding sequence; and exposing the vector to a
recombinase, wherein said recombinase recombines said first
recombination site and said second recombination to produce an
inverted coding sequence and expression of the protein of
interest.
13. The method of claim 12, wherein said exposing the vector to a
recombinase renders said inverted coding sequence incapable of
subsequent recombination.
14. The method of claim 12, wherein said first recombination site
is an attP site and said second recombination site is an attB
site.
15. The method of claim 14, wherein said attP site and said attB
site recombine in the presence of a unidirectional, site-specific
recombinase.
16. The method of claim 15, wherein said unidirectional,
site-specific recombinase is phiC31 integrase.
17. The method of claim 12, wherein said episomal circular
expression vector further comprises a third recombination site
interposed between said first recombination site and said coding
sequence and a fourth recombination site interposed between said
second recombination site and said transcription termination
region, wherein said first recombination site and said second
recombination site recombine in the presence of a recombinase and
said third recombination site and said fourth recombination site
recombine in the presence of a recombinase.
18. The method of claim 17, wherein said first recombination site
and said second recombination site are LoxP sites and said third
recombination site and said fourth recombination site are Lox2722
sites.
19. The method of claim 17, wherein said recombinase is Cre
recombinase or Flp.
20. The method of claim 18, wherein said episomal vector is an
adeno-associated vector.
21. The method of claim 18, wherein said episomal vector is an
EBNA-1 based episomal vector.
22. A kit comprising: a circular expression vector, comprising i) a
promoter; ii) a multiple cloning site for inserting a coding
sequence in a reversed 3'-5' orientation; iii) a transcription
termination sequence; and iv) at least a first recombination site
and a second recombination site flanking the coding sequence, and
instructions for using said vector.
23. The kit of claim 22, wherein said kit further comprises
cells.
24. The kit of claim 23, wherein said cells express a
recombinase.
25. The kit of claim 24, wherein said recombinase is Cre
recombinase, Flp, or phiC31 integrase.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/087,903, filed Aug. 11, 2008, which application
is incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] Genetic tools that enable researchers to control gene
expression, to delete undesired DNA sequences, and to modify
chromosome architecture have been indispensable in the advancement
of biotechnology discoveries and biomedical applications.
[0003] Precise temporal and spatial control of protein expression
in a cell or in an organism opens the door to many opportunities in
the studies of both gene expression and the physiological effects
of transgenes. As such, the ability to control the site of
integration, the number of integrated copies and the level of
expression of transgenes is very important and requires efficient
and reliable genetic tools.
[0004] In many applications, the success in regulating protein
expression depends largely on the ability to achieve a combination
of stable chromosomal integration and a tight control over the
expression of transferred genes. Certain applications utilize
drug-inducible systems and various genetic regulatory elements in
the control of gene expression in cells or transgenic animals. By
alleviating problems commonly encountered in these systems such as
leakiness, insufficient levels of induction, and lack of tissue
specificity, one may increase the in vivo functionality of these
genetic tools.
[0005] The present invention addresses these needs.
SUMMARY OF THE INVENTION
[0006] A composition for expressing a protein in cells is provided.
In certain embodiments, a circular expression vector provided
herein comprises: a promoter, a coding sequence encoding a protein
of interest, in which the coding sequence is in a reversed 3'-5'
orientation, a transcription termination sequence, and at least a
first recombination site and a second recombination site flanking
the coding sequence. A method for using the disclosed composition
and a kit comprising the composition are also provided herein.
[0007] These and other objects, advantages, and features of the
invention will become apparent to those persons skilled in the art
upon reading the details of the composition as more fully described
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] This disclosure is best understood from the following
detailed description when read in conjunction with the accompanying
drawings. It is emphasized that, according to common practice, the
various features of the drawings are not to-scale. On the contrary,
the dimensions of the various features are arbitrarily expanded or
reduced for clarity. Included in the drawings are the following
figures:
[0009] FIG. 1A schematically illustrates certain features of one
embodiment of the composition provided herein. FIG. 1B
schematically illustrates certain features of another embodiment of
the composition provided herein with a multiple cloning site. FIG.
1C schematically illustrated certain features of an additional
embodiment of the composition provided herein. FIG. 1D
schematically illustrated certain features of an additional
embodiment of the composition with a multiple cloning site.
[0010] FIG. 2A schematically illustrates certain examples of
expression cassettes. FIG. 2B presents fluorescence micrographs of
HEK293 cells in the presence or absence of Cre recombinase, as
indicated. Cells in the top panels were transfected with the Floxed
Stop construct. Cells in the middle panels were transfected with
the Single-floxed Reverse ORF (SIO) construct. Cells in the bottoms
panels were transfected with the Double-floxed Reverse ORF (DIO)
construct. Results from the FACS analysis of the various cell
populations in FIG. 2B are represented as normalized cell
population versus raw YFP fluorescence (FIG. 2C). FIG. 2D
schematically illustrates certain features of the composition and
method provided herein with an exemplary DIO construct.
[0011] FIG. 3A presents fluorescence micrographs of hippocampal
neurons transfected with DIO constructs containing a coding region
for the protein ChR2-EYFP. Middle panel monitors the presence of
parvalbumin (PV) and the right panel overlays Chr2-EYFP with PV.
Percentage of cells that are positive for either YFP or PV are
graphed in FIG. 3B. Current trace of a neuron transfected with
Chr2-EYFP construct when subjected to a light stimulus is shown in
FIG. 3C. Voltage trace of a neuron transfected with the Chr2-EYFP
construct when subjected to pulses of light stimulus is shown in
FIG. 3D.
[0012] FIG. 4A depicts several exemplary microbial opsins that may
be used in the subject method and their electrophysiological
activities in response to light.
[0013] FIG. 4B depicts exemplary strategies in utilizing
anterograde and retrograde transport for selective gene
expression.
[0014] FIG. 5A is a schematic illustrating gene expression
targeting using anterograde transport. FIG. 5B is a schematic
illustrating gene expression targeting using retrograde transport.
FIG. 5C depicts construct design for the WGA-CRE and Cre-TTC
adeno-associated virus vectors used in FIGS. 5A and 5B,
respectively. FIG. 5D shows fluorescence images demonstrating the
activation of Cre-dependent gene expression in the contralateral
dentate gyms and the Cre-expression of the virus vectors in the
ipsilateral dentate gyms.
[0015] FIG. 6 summarizes nine exemplary strategies for controlling
a specific neural component in one of the nine given networks, each
of which comprises cell population A, cell population B, and cell
population C.
DEFINITIONS
[0016] The terms "nucleic acid molecule" and "polynucleotide" are
used interchangeably and refer to a polymeric form of nucleotides
of any length, either deoxyribonucleotides or ribonucleotides, or
analogs thereof. Polynucleotides may have any three-dimensional
structure, and may perform any function, known or unknown.
Non-limiting examples of polynucleotides include a gene, a gene
fragment, exons, introns, messenger RNA (mRNA), transfer RNA,
ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides,
branched polynucleotides, plasmids, vectors, isolated DNA of any
sequence, control regions, isolated RNA of any sequence, nucleic
acid probes, and primers. The nucleic acid molecule may be linear
or circular.
[0017] A polynucleotide is typically composed of a specific
sequence of four nucleotide bases: adenine (A); cytosine (C);
guanine (G); and thymine (T), (uracil (U) for thymine (T) when the
polynucleotide is RNA). Thus, the term polynucleotide sequence is
the alphabetical representation of a polynucleotide molecule. This
alphabetical representation can be input into databases in a
computer having a central processing unit and used for
bioinformatics applications such as functional genomics and
homology searching.
[0018] A "polypeptide" is a sequence or a portion thereof that
contains an amino acid sequence of at least 3 to 5 amino acids,
more preferably at least 8 to 10 amino acids, and even more
preferably at least 15 to 20 amino acids. A polypeptide may be
encoded by a nucleic acid sequence. Also encompassed are
polypeptide sequences that are immunologically identifiable with a
polypeptide encoded by the sequence.
[0019] A "coding sequence" or a sequence that "encodes" a selected
polypeptide, is a nucleic acid molecule which is transcribed (in
the case of DNA) and translated (in the case of mRNA) into a
polypeptide, for example, in vivo when placed under the control of
appropriate regulatory sequences (or "control elements"). The
boundaries of the coding sequence are typically determined by a
start codon at the 5' (amino) terminus and a translation stop codon
at the 3' (carboxy) terminus. A coding sequence can include, but is
not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,
genomic DNA sequences from viral or prokaryotic DNA, and even
synthetic DNA sequences. A transcription termination sequence may
be located 3' to the coding sequence. Other "control elements" may
also be associated with a coding sequence. A DNA sequence encoding
a polypeptide can be optimized for expression in a selected cell by
using the codons preferred by the selected cell to represent the
DNA copy of the desired polypeptide coding sequence.
[0020] As used herein, the term "gene" or "recombinant gene" refers
to a nucleic acid comprising an open reading frame encoding a
polypeptide of the present invention, including both exon and
(optionally) intron sequences. A "recombinant gene" refers to
nucleic acid encoding such regulatory polypeptides, that may
optionally include intron sequences derived from chromosomal
DNA.
[0021] As used here, the term "inverted" or "inversion" refers to a
nucleic acid sequence that is in an opposite orientation specified
by its 5' and 3' ends relative to its original orientation in the
context of a genome or a longer polynucleotide. In certain
embodiments, 3' to 5' coding sequence may be referred herein as in
the "reversed 3' to 5' orientation" due to the convention of
annotating nucleic acid elements in the 5' to 3' direction.
[0022] The term "promoter" or "promoter element" is defined herein
as a nucleic acid that directs transcription of a downstream
polynucleotide in a cell. In certain cases, the polynucleotide may
contain a coding sequence and the promoter may direct the
transcription of the coding sequence into translatable RNA.
[0023] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, a given promoter that is operably
linked to a coding sequence (e.g., a reporter expression cassette)
is capable of effecting the expression of the coding sequence when
the proper enzymes are present. The promoter or other control
elements need not be contiguous with the coding sequence, so long
as they function to direct the expression thereof. For example,
intervening untranslated yet transcribed sequences can be present
between the promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence.
[0024] By "nucleic acid construct," it is meant a nucleic acid
sequence that has been constructed to comprise one or more
functional units not found together in nature. Examples include
circular, linear, double-stranded, extrachromosomal DNA molecules
(plasmids), cosmids (plasmids containing COS sequences from lambda
phage), viral genomes comprising non-native nucleic acid sequences,
and the like.
[0025] A "vector" is capable of transferring gene sequences to
target cells. Typically, "vector construct," "expression vector,"
and "gene transfer vector," mean any nucleic acid construct capable
of directing the expression of a gene of interest and which can
transfer gene sequences to target cells. Thus, the term includes
cloning and expression vehicles, as well as integrating
vectors.
[0026] The term "expression" with respect to a gene sequence refers
to transcription of the gene and, as appropriate, translation of
the resulting mRNA transcript to a protein. Thus, as will be clear
from the context, expression of a protein coding sequence results
from transcription and translation of the coding sequence.
[0027] An "expression cassette" comprises any nucleic acid
construct capable of directing the expression of a gene/coding
sequence of interest. Such cassettes can be constructed into a
"vector," "vector construct," "expression vector," or "gene
transfer vector," in order to transfer the expression cassette into
target cells. Thus, the term includes cloning and expression
vehicles, as well as viral vectors. Expression cassettes include at
least promoters and optionally, transcription termination signals.
Additional factors necessary or helpful in effecting expression can
also be used as described herein. For example, transcription
termination signals, enhancers, and other nucleic acid sequences
that influence gene expression, can also be included in an
expression cassette.
[0028] The term "episomal vector," as used herein, refers to a
vector introduced into the target cells that does not integrate
into, i.e., insert into, the target cell genome, i.e., one or more
chromosomes of the target cell. In other words, an episomal vector
does not fuse with or become covalently attached to chromosomes
present in the target cell into which it is introduced.
Accordingly, episomal vectors provide for persistent expression,
while being maintained episomally.
[0029] The term "exogenous" is defined herein as DNA which is
introduced into a cell. Exogenous DNA can possess sequences
identical to or different from the endogenous DNA present in the
cell prior to transfection.
[0030] As used herein, "transgene" or "transgenic element" refers
to an artificially introduced, chromosomally integrated nucleic
acid sequence present in the genome of a host organism.
[0031] The term "transgenic animal" means a non-human animal having
a transgenic element integrated in the genome of one or more cells
of the animal. "Transgenic animals" as used herein thus encompasses
animals having all or nearly all cells containing a genetic
modification (e.g., fully transgenic animals, particularly
transgenic animals having a heritable transgene) as well as
chimeric, transgenic animals, in which a subset of cells of the
animal are modified to contain the genomically integrated
transgene.
[0032] "Target cell" as used herein refers to a cell that in which
a genetic modification is desired. Target cells can be isolated
(e.g., in culture) or in a multicellular organism (e.g., in a
blastocyst, in a fetus, in a postnatal animal, and the like).
Target cells of particular interest in the present application
include, but not limited to, cultured mammalian cells, including
CHO cells, and stem cells (e.g., embryonic stem cells (e.g., cells
having an embryonic stem cell phenotype), adult stem cells,
pluripotent stem cells, hematopoietic stem cells, mesenchymal stem
cells, and the like).
[0033] "Recombinases" are a family of enzymes that mediate
site-specific recombination between specific DNA sequences
recognized by the recombinase (Esposito, D., and Scocca, J. J.,
Nucleic Acids Research 25, 3605-3614 (1997); Nunes-Duby, S. E., et
al., Nucleic Acids Research 26, 391-406 (1998); Stark, W. M., et
al., Trends in Genetics 8, 432-439 (1992)). Within this group are
several subfamilies including "Integrase" or tyrosine recombinase
(including, for example, Cre and .lamda. integrase) and
"Resolvase/Invertase" or serine recombinase (including, for
example, .phi.C31 integrase, R4 integrase, and TP-901 integrase).
The term also includes recombinases that are altered as compared to
wild-type, for example as described in U.S. Patent Publication
20020094516, the disclosure of which is hereby incorporated by
reference in its entirety herein.
[0034] A "unidirectional site-specific recombinase" is a
naturally-occurring recombinase, such as the .phi.C31 integrase, a
mutated or altered recombinase, such as a mutated or altered
.phi.C31 integrase that retains unidirectional, site-specific
recombination activity, or a bi-directional recombinase modified so
as to be unidirectional, such as a Cre recombinase that has been
modified to become unidirectional.
[0035] "Site-specific integration" or "site-specifically
integrating" as used herein refers to the sequence specific
recombination and integration of a first nucleic acid with a second
nucleic acid, typically mediated by a recombinase. In general,
site-specific recombination or integration occurs at particular
defined sequences recognized by the recombinase. These defined
sequences are referred herein as "recombination sites." In contrast
to random integration, site specific integration occurs at a
particular sequence at a higher efficiency.
[0036] The term "recombination site," as used herein, refers to a
nucleotide sequence recognized by a recombinase so that a
recombination event may occur. In certain cases, the recombination
sites may be recognizable by Cre, Flp, integrase, or other
recombinase. An example of a recombination site recognizable by the
Cre recombinase is loxP, a sequence of about 34 base pairs
comprising an .about.8 base pair core sequence flanked by two
.about.13 base pair inverted repeats (Sauer, Curr. Opin. Biotech.
5:521-527, 1994). In certain embodiments, the recombination sites
are sequences between short (about 15 to about 40 base pair)
Flipase Recognition Target (FRT) sites, recognizable by the Flipase
recombination enzyme (FLP or Flp) derived from the yeast
Saccharomyces cerevisiae (U.S. Pat. No. 5,527,695, Lyznik et al.
Nucleic Acid Res. 24:3784-3789, 1996, and O'Gorman et al., Science,
251:1351-1355, 1991). Another recombination sites may be attP or
attB attachment site sequences that are recognizable by phage
.phi.C31.
[0037] A "native recognition site", as used herein, means a
recognition site that occurs naturally in the genome of a cell
(i.e., the sites are not introduced into the genome, for example,
by recombinant means).
[0038] A "pseudo-site" or a "pseudo-recombination site" as used
herein means a DNA sequence comprising a recognition site that is
bound by a recombinase enzyme where the recognition site differs in
one or more nucleotides from a wild-type recombinase recognition
sequence and/or is present as an endogenous sequence in a genome
that differs from the sequence of a genome where the wild-type
recognition sequence for the recombinase resides. For a given
recombinase, a pseudo-recombination sequence is functionally
equivalent to a wild-type recombination sequence, occurs in an
organism other than that in which the recombinase is found in
nature, and may have sequence variation relative to the wild type
recombination sequences. In some embodiments a "pseudo attP site"
or "pseudo attB site" refer to pseudo sites that are similar to the
recognitions site for wild-type phage (attP) or bacterial (attB)
attachment site sequences, respectively, for phage integrase
enzymes, such as the phage .phi.C31.
[0039] Methods of transfecting cells are well known in the art. By
"transfected" it is meant an alteration in a cell resulting from
the uptake of foreign nucleic acid, usually DNA. Use of the term
"transfection" is not intended to limit introduction of the foreign
nucleic acid to any particular method. Suitable methods include
viral infection, conjugation, electroporation, particle gun
technology, calcium phosphate precipitation, direct microinjection,
and the like. The choice of method is generally dependent on the
type of cell being transfected and the circumstances under which
the transfection is taking place (i.e. in vitro, ex vivo, or in
vivo). A general discussion of these methods can be found in
Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed.,
Wiley & Sons, 1995.
DETAILED DESCRIPTION OF THE INVENTION
[0040] A composition for expressing a protein in cells is provided.
In certain embodiments, a circular expression vector provided
herein comprises: a promoter, a coding sequence encoding a protein
of interest, in which the coding sequence is in a reversed 3'-5'
orientation, a transcription termination sequence, and at least a
first recombination site and a second recombination site flanking
the coding sequence.
[0041] Before the present composition are described, it is to be
understood that this invention is not limited to particular
composition described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0042] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0043] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supercedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0044] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a vector" includes a plurality of such
vectors and reference to "the protein" includes reference to one or
more proteins and equivalents thereof known to those skilled in the
art, and so forth.
[0045] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Compositions
[0046] As noted above, in certain embodiments, the composition
provided herein comprises a circular expression vector comprising:
a promoter, a coding sequence encoding a protein of interest, in
which the coding sequence is in a reversed 3'-5' orientation, a
transcription termination sequence, and at least a first
recombination site and a second recombination site flanking the
coding sequence.
[0047] Certain features of the subject composition are illustrated
in FIG. 1 and are described in greater detail below. With reference
to FIG. 1, the circular expression vector 2 comprises the following
elements in order from 5' to 3', a promoter sequence 4, a first
recombination site 6, a coding sequence 8, a second recombination
site 12, and a transcription termination sequence 18.
[0048] In certain embodiments, the coding sequence is oriented in
the reversed 3' to 5', in contrast to the rest of the nucleic acid
elements in the vector. In other words, the coding sequence is
positioned in the antisense orientation with regard to the
promoter. This reversed position prevents the expression of the
coding sequence in the presence of a functional machinery for
transcription or translation because the sequence is not in the
correct orientation for transcription or translation to proceed.
For example, a transcript made from such reversed coding sequence
may lack a proper start codon or may not in the right frame, giving
rise to a premature stop codon.
[0049] Generally, the coding sequence is positioned in an
orientation such that a product would express only if the
orientation is inverted. In certain embodiments, the first and
second recombination sites flanking the coding sequence are
recognized by a recombinase that causes the coding sequence to
undergo recombination, and consequently inverting the orientation
of the coding sequence. A feature of the invention is that the
product resulting from a recombination reaction is incapable of
undergoing subsequence recombination. Hence, once the coding
sequence in the reversed 3' to 5' orientation undergoes a
recombination, the sequence becomes inverted and stays in the 5' to
3' orientation for the lifetime of the vector. The fact that the
product of the recombination reaction is incapable of further
recombination means that the recombined vector permanently
comprises a coding sequence that is capable of being expressed. The
coding sequence would not be excised out or recombined back into
the reversed 3' to 5' position.
[0050] In certain embodiments, with reference to FIG. 1, a first
and a second recombination sites (6 and 12) may be a phage
attachment site ("attP") and a bacterial attachment site ("attB"),
respectively. The native attB and attP recognition sites of phage
.phi.C31 (i.e. bacteriophage .phi.C31) are generally about 34 to 40
nucleotides in length (Groth et al. Proc Natl Acad Sci USA
97:5995-6000 (2000)). These sites are typically arranged as
follows: attP comprises a first DNA sequence (attP5'), a core
region, and a second DNA sequence (attP3') in the relative order
attP5'-core region-attP3'; AttB comprises a first DNA sequence
attB5', a core region, and a second DNA sequence attB3' in the
relative order attB5'-core region-attB3'.
[0051] For example, for the phage .phi.C31 attP (the phage
attachment site), the core region is 5'-TTG-3' the flanking
sequences on either side are represented here as attP5' and attP3',
the structure of the attP recombination site is, accordingly,
attP51-TTG-attP3'. Correspondingly, for the native bacterial
genomic target site (attB) the core region is 5'-TTG-3', and the
flanking sequences on either side are represented here as attB5 `
and attB3', the structure of the attB recombination site is,
accordingly, attB51-TTG-attB3`.
[0052] Because the attB and attP sites are different sequences,
recombination results in a hybrid site-specific recombination site
(designated attL or attR for left and right) that is neither an
attB sequence or an attP sequence, and is functionally
unrecognizable as a site-specific recombination site (e.g., attB or
attP) to the relevant unidirectional site-specific recombinase,
thus removing the possibility that the unidirectional site-specific
recombinase will catalyze a second recombination reaction between
the attL and the attR, reversing the first recombination reaction.
For example, after a single-site, .phi.C31 integrase-mediated,
recombination event takes place the result is the following
recombination product: attB5'-TTG-attP3'{.phi.C31 vector
sequences}attP5'-TTG-attB3'. Typically, after recombination the
post-recombination recombination sites are no longer able to act as
substrate for the .phi.C31 recombinase. This results in stable
integration with little or no recombinase mediated excision.
[0053] In certain cases, the recombination sites in the subject
vector may be native recombination sites, found to exist in the
genomes of a variety of organisms. The native recombination site
does not necessarily have a nucleotide sequence identical to the
wild-type recombination sequences (for a given recombinase); but
such native recombination sites are nonetheless sufficient to
promote recombination meditated by the recombinase. Such
recombination site sequences are referred to herein as
"pseudo-recombination sequences." For a given recombinase, a
pseudo-recombination sequence is functionally equivalent to a
wild-type recombination sequence, occurs in an organism other than
that in which the recombinase is found in nature, and may have
sequence variation relative to the wild type recombination
sequences.
[0054] Identification of pseudo-recombination sequences can be
accomplished, for example, by using sequence alignment and
analysis, where the query sequence is the recombination site of
interest (for example, attP and/or attB).
[0055] In general, the unidirectional site-specific integrase
interaction with the site-specific recombination sites produces a
recombination product that does not contain a sequence that acts as
an effective substrate for the unidirectional site-specific
integrase. Thus, the recombination event occurring with the subject
composition is unidirectional, with little or no detectable
excision of the introduced nucleic acid mediated by the
unidirectional site-specific integrase.
[0056] In addition, to a first and a second recombination sites, in
certain embodiments, the subject composition may further comprise a
third recombination site interposed between the first recombination
site and the coding sequence and a fourth recombination site
interposed between the second recombination site and the
transcription termination region, wherein the first recombination
site and the second recombination site recombine in the presence of
a recombinase and the third recombination site and the fourth
recombination site recombine in the presence of a recombinase.
[0057] With reference to FIG. 1C, the subject composition according
to the above embodiment is an expression vector comprising, in an
order from 5' to 3': a promoter 4, a first recombination site 6, a
third recombination site 14, a coding sequence in a reversed 3' to
5' orientation 8, a second recombination site 12, a fourth
recombination site 16, followed by a transcription termination
sequence 18.
[0058] A vector where there are two recombination sites flanking
each side of the coding sequence may undergo a recombination event
to invert the coding sequence into a 5' to 3' orientation to turn
on expression. After such a recombination event, two of the four
existing recombination sites may be excised in a subsequent
recombination to produce a vector with one recombination site
flanking each side of the coding sequence. This two-step
recombination event produces a vector that is incapable of
subsequent recombination and the coding sequence remains in the 5'
to 3' orientation.
[0059] In certain cases, with reference to FIG. 1C, the first
recombination site 6 and the second recombination site 12 form a
first pair of compatible sites such that a recombinase would
recognize the first and second sites to catalyze a recombination
event. Accordingly, the third and the fourth recombination sites
(14 and 16) form a second pair of compatible sites such that a
recombination event may occur based on the recognition of these
compatible sites by a recombinase. In certain embodiments, a
recombination site from a first pair is not compatible with either
site in a second pair. A recombinase is not able to catalyze a
recombination event to excise the coding sequence or to invert the
coding sequence if the only recombination sites flanking the coding
sequence are incompatible.
[0060] As such, in an embodiment where a vector comprises four
recombination sites, as described above, the subject vector may
lose compatible recombination sites after a two-step recombination
event, rendering the coding sequence incapable of subsequence
recombination.
[0061] In certain cases, the recombination sites in the subject
vector may be recognizable by a Cre, Flp, or other recombinase. For
example, the recognition sequence for Cre recombinase is loxP which
is a sequence of about 34 base pairs comprising an 8 base pair core
sequence flanked by two 13 base pair inverted repeats (serving as
the recombinase binding sites) (Sauer, Curr. Opin. Biotech.
5:521-527, 1994). In other embodiments, the recognition sequence
for Cre recombinase is lox2722, which contain certain mutations
that render the site incompatible with loxP. Other incompatible
recombination sites may also be used.
[0062] In certain embodiments, the recombination sites are
sequences between short (about 15 to about 40 base pair) Flipase
Recognition Target (FRT) sites, recognizable by the Flipase
recombination enzyme (FLP or Flp) derived from the yeast
Saccharomyces cerevisiae (U.S. Pat. No. 5,527,695, Lyznik et al.
Nucleic Acid Res. 24:3784-3789, 1996, and O'Gorman et al., Science,
251:1351-1355, 1991).
[0063] Other examples of recognition sequences are the attB, attP,
attL, and attR sequences, as described above, which are recognized
by the recombinase enzyme .lamda. Integrase. AttB is an
approximately 25 base pair sequence containing two 9 base pair
core-type Int binding sites and a 7 base pair overlap region. AttP
is an approximately 240 base pair sequence containing core-type Int
binding sites and arm-type Int binding sites as well as sites for
auxiliary proteins IHF, FIS, and Xis. See Landy, Curr. Opin.
Biotech. 3:699-707 (1993). Other phage integrases (such as the R4
phage integrase) and their recognition sequences can be adapted for
use in the invention.
[0064] In certain cases, a pair of compatible loxP sites is
positioned in the reversed orientation with respect to one another
so that recombination between the two sites within the same
polynucleotide leads to an inversion of the intervening sequences,
as opposed to excision. For example, sites 6 and 12 in FIG. 1 may
be compatible loxP sites that are positioned in the reversed
orientation with respect to one another. Recombination between site
6 and 12 leads to inversion of the coding sequence 8. In certain
embodiments, the inversion of the coding sequence leads to one pair
of compatible recombination sites on one side of the coding
sequence in the same orientation, as opposed to the reversed
orientation with respect to one another. Such position of the
recombination sites allows subsequent excision of the intervening
sequence, leaving the coding sequence flanked by incompatible
sites. Accordingly, the coding sequence may not undergo another
recombination event due to the absence of compatible recombination
sites.
[0065] Certain features of this embodiment are illustrated in an
exemplary expression cassette in FIG. 2D. The expression cassette
may be present in the context of a larger polynucleotide, such as a
vector of the subject invention. In an order from 5' to 3', the
cassette contains a promoter, such as EF-1a, one loxP site, one
lox2722 site, followed by an exemplary coding sequence labeled as
ChR2-EYFP in a reversed orientation, and a loxP site, followed by
another lox2722 site. Following a recombination event, the coding
sequence is inverted to be in an orientation enabling correct
transcription and translation. One pair of compatible recombination
sites are also rearranged to be either on the 5' side or the 3'
side of ChR2-EYFP, depending on which pair of recombination sites
is used by the recombinase. For example, in the middle top panel,
both lox2722 sites are located on the 3' side of ChR2-EYFP, as
directed repeats, as opposed to the inverted repeats that have
existed before the recombination event. A different scenario is
illustrated by the bottom panel, where both loxP sites may be
located on the 5' side of ChR2-EYFP, also as directed repeats. The
orientation of the directed repeats leads to an excision of the
intervening sequence by the recombinase. The final product is an
expression cassette in the right panel containing: in an order from
5' to 3', a promoter EF-1a, a loxP site, ChR2-EYFP, followed by a
lox2722 site. Since loxP and lox2722 are incompatible with each
other, their intervening sequence, namely the coding sequence
ChR2-EYFP, is incapable of subsequent recombination.
[0066] This feature of the subject composition ensures stability of
expression of proteins of interest once a recombination event has
taken place to invert the coding sequence to a translatable 5' to
3' orientation.
[0067] In certain embodiments, the subject composition may also
comprise selectable markers, an origin of replication, and other
elements such as an inducible element sequence, an epitope tag
sequence, a promoter, or promoter-enhancer sequences, and the like.
See, e.g., U.S. Pat. No. 6,632,672, the disclosure of which is
incorporated by reference herein in its entirety. The promoter
element is discussed in more detail below.
[0068] The promoter sequence is operably linked to the coding
sequence as to promote the transcription of the coding sequence
when the appropriate enzymes are present. Promoter and
promoter-enhancer sequences are DNA sequences to which RNA
polymerase binds and initiates transcription. The promoter
determines the polarity of the transcript by specifying which
strand will be transcribed. Bacterial promoters consist of
consensus sequences, -35 and -10 nucleotides relative to the
transcriptional start, which are bound by a specific sigma factor
and RNA polymerase.
[0069] Eukaryotic promoters are more complex. Most eukaryotic
promoters utilized in expression vectors are transcribed by RNA
polymerase II. General transcription factors (GTFS) first bind
specific sequences near the transcription start site and then
recruit the binding of RNA polymerase II. In addition to these
minimal promoter elements, small sequence elements are recognized
specifically by modular DNA-binding, trans-activating proteins
(e.g. AP-1, SP-1) that regulate the activity of a given promoter.
Viral promoters serve the same function as bacterial or eukaryotic
promoters and either require a promoter-specific RNA polymerase in
trans (e.g., bacteriophage T7 RNA polymerase in bacteria) or
recruit cellular factors and RNA polymerase II (in eukaryotic
cells). Viral promoters (e.g., the SV40, RSV, and CMV promoters)
may be preferred as they are generally particularly strong
promoters.
[0070] Promoters may be, furthermore, either constitutive or
regulatable. Constitutive promoters constantly express the gene of
interest. In contrast, regulatable promoters (i.e., derepressible
or inducible) express genes of interest only under certain
conditions that can be controlled. Derepressible elements are DNA
sequence elements which act in conjunction with promoters and bind
repressors (e.g. lacO/lacIq repressor system in E. coli). Inducible
elements are DNA sequence elements which act in conjunction with
promoters and bind inducers (e.g. gal1/gal4 inducer system in
yeast). In either case, transcription is virtually "shut off" until
the promoter is derepressed or induced by alteration of a condition
in the environment (e.g., addition of IPTG to the lacO/lacIq system
or addition of galactose to the gal1/gal4 system), at which point
transcription is "turned-on."
[0071] Another type of regulated promoter is a "repressible" one in
which a gene is expressed initially and can then be turned off by
altering an environmental condition. In repressible systems
transcription is constitutively on until the repressor binds a
small regulatory molecule at which point transcription is "turned
off". An example of this type of promoter is the
tetracycline/tetracycline repressor system. In this system when
tetracycline binds to the tetracycline repressor, the repressor
binds to a DNA element in the promoter and turns off gene
expression.
[0072] Examples of constitutive prokaryotic promoters include the
int promoter of bacteriophage .lamda., the bla promoter of the
.beta.-lactamase gene sequence of pBR322, the CAT promoter of the
chloramphenicol acetyl transferase gene sequence of pPR325, and the
like.
[0073] Examples of inducible prokaryotic promoters include the
major right and left promoters of bacteriophage (P.sub.L and
P.sub.R), the trp, recA, lacZ, AraC and gal promoters of E. coli,
the .alpha.-amylase (Ulmanen Ett at., J. Bacteriol. 162:176-182,
1985) and the sigma-28-specific promoters of B. subtilis (Gilman et
al., Gene sequence 32:11-20 (1984)), the promoters of the
bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of
the Bacilli, Academic Press, Inc., NY (1982)), Streptomyces
promoters (Ward et at., Mol. Gen. Genet. 203:468-478, 1986), and
the like. Exemplary prokaryotic promoters are reviewed by Glick (J.
Ind. Microtiot. 1:277-282, 1987); Cenatiempo (Biochimie 68:505-516,
1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).
[0074] Exemplary constitutive eukaryotic promoters include, but are
not limited to, the following: the promoter of the mouse
metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.
1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell
31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature
(London) 290:304-310, 1981); the yeast gal1 gene sequence promoter
(Johnston et al., Proc. Natl. Acad. Sci. USA 79:6971-6975, 1982);
Silver et al., Proc. Natl. Acad. Sci. USA 81:5951-59SS, 1984), the
CMV promoter, the EF-1 promoter.
[0075] Examples of inducible eukaryotic promoters include, but are
not limited to, the following: ecdysone-responsive promoters, the
tetracycline-responsive promoter, promoters regulated by
"dimerizers" that bring two parts of a transcription factor
together, estrogen-responsive promoters, progesterone-responsive
promoters, riboswitch-regulated promoters, antibiotic-regulated
promoters, acetaldehyde-regulated promoters, and the like.
[0076] Some regulated promoters can mediate both repression and
activation. For example, in the RheoSwitch system a protein (the
RheoReceptor) binds to a DNA element (UAS, upstream activating
sequence) in the promoter and mediates repression. However in the
presence of certain ecdysone-like inducers another protein (the
RheoActivator) will bind to the inducer. The inducer-bound
RheoActivator is capable of binding to the DNA-bound RheoReceptor.
The RheoReceptor/inducer/RheoActivator is then capable of
activating gene expression.
[0077] As noted above, in certain embodiments, the subject
composition also comprises selectable markers. Common selectable
marker genes include those for resistance to antibiotics such as
ampicillin, tetracycline, kanamycin, bleomycin, streptomycin,
hygromycin, neomycin, puromycin, G418, bleomycin, blasticidin,
Zeocin.TM., and the like. Selectable auxotrophic genes include, for
example, hisD, that allows growth in histidine free media in the
presence of histidinol.
[0078] A further element useful in an expression vector is an
origin of replication. Replication origins are unique DNA segments
that contain multiple short repeated sequences that are recognized
by multimeric origin-binding proteins and that play a key role in
assembling DNA replication enzymes at the origin site. Suitable
origins of replication for use in expression vectors employed
herein include E. coli oriC, ColE1 plasmid origin, 2.mu. and ARS
(both useful in yeast systems), sf1, SV40, EBV oriP (useful in
eukaryotic systems, such as a mammalian system), and the like.
[0079] In certain cases, the subject vector is an episomal vector.
In certain embodiments, the composition contains an Epstein Barr
virus (EBV) oriP origin of replication, which permits episomal
replication in cell lines expressing EBV nuclear antigen (EBNA-1).
Vectors having the EBV origin and the nuclear antigen EBNA-1 are
capable of replication to high copy number in mammalian cells
without being integrated in the genome of the cell. In certain
embodiments, the presence of EBNA-1 in combination with the OriP
latent origin of replication, confer the functions of autonomous
episomal replication and nuclear retention in a stable copy number,
replicating only once per cell cycle.
[0080] In certain embodiments, the subject composition is derived
from adenovirus or comprises adenovirus-associated components. For
example, an episomal vector may be characterized by the following
features: (i) an element, such as the EBV plasmid origin of
replication, which renders the episome capable of autonomous
replication and maintains the episome in multiple copies by
promoting nuclear retention, (ii) an adenoviral type Ad5 inverted
terminal repeat (ITRs) junction; and (iii) elements mediating the
expression of adenoviral genes necessary for adenoviral replication
(e.g., polymerase, pre-terminal protein and DNA binding protein, as
well as early region 4 (E4) ORF6. Details of the adenoviral genomes
and the methods of using adenovirus-associated vectors are
discussed in U.S. Pat. Nos. 6,303,362 and 7,045,344, disclosures of
which are incorporated herein by reference.
[0081] In certain embodiments, the subject composition further
comprises an internal ribosome entry site (IRES) positioned in the
coding sequence between the transcription start site and the
translation initiation codon of the protein of interest. Such
vectors may allow for increased gene expression if they are
translational enhancers or they can also allow for production of
multiple proteins of interest from a single transcript, as long as
an IRES is located 5' to each coding region of interest.
[0082] In certain cases, the subject composition includes a
multiple cloning site or polylinker A multiple cloning site or
polylinker is a synthetic DNA encoding a series of restriction
endonuclease recognition sites inserted into the subject vector and
allows for convenient cloning of polynucleotides of interest into
the donor vector at a specific position. By "polynucleotide of
interest" it is meant any nucleic acid fragment adapted for
introduction into a target cell. Suitable examples of
polynucleotides of interest include promoter elements, therapeutic
genes, marker genes, control regions, trait-producing fragments,
nucleic acid elements to encode a polypeptide, gene disruption
elements, as well as nucleic acids that do not encode for a
polypeptide, including a polynucleotide that encodes a
non-translated RNA, such as a shRNA that may play a role in RNA
interference (RNAi) based gene expression control. FIGS. 1B and 1D
schematically illustrate certain features of an embodiment of the
subject composition comprising a multiple cloning site 10. In
certain cases, the multiple cloning site 10 enables the insertion
of a coding sequence in the reversed 3' to 5' orientation relative
to the promoter 4.
[0083] The subject composition described herein can be constructed
utilizing methodologies known in the art of molecular biology (see,
for example, Ausubel or Maniatis) in view of the teachings of the
specification. An exemplary method of obtaining polynucleotides,
including suitable regulatory sequences (e.g., promoters) is PCR.
General procedures for PCR are taught in MacPherson et al., PCR: A
PRACTICAL APPROACH, (IRL Press at Oxford University Press, (1991)).
PCR conditions for each application reaction may be empirically
determined A number of parameters influence the success of a
reaction. Among these parameters are annealing temperature and
time, extension time, Mg.sup.2+ and ATP concentration, pH, and the
relative concentration of primers, templates and
deoxyribonucleotides. After amplification, the resulting fragments
can be detected by agarose gel electrophoresis followed by
visualization with ethidium bromide staining and ultraviolet
illumination.
[0084] In certain embodiments, the present disclosure further
provides cells containing an expression vector, as described above,
that contains a promoter; a coding sequence encoding a protein of
interest in a reversed 3'-5' orientation; a transcription
termination sequence; and at least a first recombination site and a
second recombination site flanking the coding sequence. In certain
cases, the cells may contain additional vectors or genetic elements
integrated into the cell genomes. In certain cases, the cells may
be infected with a virus, e.g. adenovirus. In certain cases, the
cells express a recombinase that recognizes the recombination sites
in the expression vector.
Methods
[0085] In certain aspects, the present disclosure provides a method
of protein expression in a cell. In certain embodiments, the method
involves transfecting a cell with a vector comprising a promoter, a
coding sequence encoding a protein of interest, in which the coding
sequence is in a reversed 3'-5' orientation, a transcription
termination sequence, and at least a first recombination site and a
second recombination site flanking the coding sequence; exposing
the vector to a recombinase, in which the recombinase recombines
the first recombination site and the second recombination site to
produce an inverted coding sequence and expresses the protein of
interest.
[0086] In certain embodiments, the method involves 1) inserting a
coding sequence encoding a protein of interest into a multiple
cloning site such that the coding sequence is in the reversed 3' to
5' orientation relative to the promoter and flanked by at least two
recombination sites, prior to transfecting the expression vector
inserted with the coding sequence into a cell.
[0087] The method of transfection is well-known in the art and may
comprise non-viral delivery systems or viral delivery systems.
Non-viral delivery systems include but are not limited to DNA
transfection methods. Here, transfection may include a process
using a non-viral vector to deliver a gene to a target mammalian
cell. Typical transfection methods include electroporation, DNA
biolistics, lipid-mediated transfection, compacted DNA-mediated
transfection, liposomes, immunoliposomes, lipofectin, cationic
agent-mediated, cationic facial amphiphiles (CFAs) (Nature
Biotechnology 1996 14; 556), and combinations thereof.
[0088] Viral delivery systems include but are not limited to
adenovirus vector, an adeno-associated viral (AAV) vector, a herpes
viral vector, retroviral vector, lentiviral vector, baculoviral
vector. In certain cases, viral based transformation protocols have
been developed to introduce exogenous DNA to be subsequently
integrated into the target cell's genome. In certain cases, the
viral vectors are maintained episomally inside a cell. Other viral
based vectors that find use include adenovirus derived vectors, HSV
derived vectors, sindbis derived vectors, and retroviral vectors,
e.g., Moloney murine leukemia viral based vectors, etc.
[0089] Other examples of vectors include ex vivo delivery
systems--which include but are not limited to DNA transfection
methods such as electroporation, DNA biolistics, lipid-mediated
transfection, compacted DNA-mediated transfection). Certain
features of the adenoviruses may be combined with the genetic
stability of retroviruses/lentiviruses to transduce target cells
that become capable of stably infect neighboring cells. Methods of
viral delivery are well-known and are described in U.S. Pat. Pub.
No. 2008/0175819, 2008/0050770, U.S. Pat. Nos. 6,303,362 and
7,045,344, disclosures of which are incorporated herein by
reference.
[0090] In certain embodiments, the subject method comprises
exposing the subject composition to a recombinase, such that the
recombinase inverts the coding sequence between the first and the
second recombination sites. The inverted coding sequence then
becomes in the correct 5' to 3' orientation to enable transcription
and translation of the expression product. In certain cases, the
inverted coding sequence is incapable of subsequent recombination
after being inverted into the 5' to 3' orientation.
[0091] Many recombinases may be used in the subject method. Two
major families of site-specific recombinases from bacteria and
unicellular yeasts have been described: the integrase or tyrosine
recombinase family includes Cre, Flp, R, and .lamda. integrase
(Argos, et al., EMBO J. 5:433-440, (1986)) and the
resolvase/invertase or serine recombinase family that includes some
phage integrases, such as, those of phages .phi.C31, R4, and
TP901-1 (Hallet and Sherratt, FEMS Microbiol. Rev. 21:157-178
(1997)). For further description of suitable site-specific
recombinases, see U.S. Pat. No. 6,632,672 and U.S. Patent
Publication No. 2003/0050258, the disclosures of which are herein
incorporated herein by reference in their entireties.
[0092] Action of the integrase upon these recognitions sites is
unidirectional in that the enzymatic reaction produces nucleic acid
recombination products that are not effective substrates of the
integrase. This results in stable integration with little or no
detectable recombinase-mediated excision, i.e., recombination that
is "unidirectional".
[0093] In certain embodiments, the recombinase used in the subject
method is a unidirectional site-specific recombinase, such as a
serine integrase. Serine integrases that may be useful for in vitro
and in vivo recombination include, but are not limited to,
integrases from phages .phi.C31, R4, TP901-1, phiBT1, Bxb1, RV-1,
A118, U153, and phiFC1, as well as others in the large serine
integrase family (Gregory, Till and Smith, J. Bacteriol.,
185:5320-5323 (2003); Groth and Calos, J. Mol. Biol. 335:667-678
(2004); Groth et al. PNAS 97:5995-6000 (2000); Olivares, Hollis and
Calos, Gene 278:167-176 (2001); Smith and Thorpe, Molec.
Microbiol., 4:122-129 (2002); Stoll, Ginsberg and Calos, J.
Bacteriol., 184:3657-3663 (2002)). In addition to these wild-type
integrases, altered integrases that bear mutations have been
produced (Sclimenti, Thyagarajan and Calos, NAR, 29:5044-5051
(2001)). These integrases may have altered activity or specificity
compared to the wild-type and are also useful for the in vitro
recombination reaction and the integration reaction into the
eukaryotic genome.
[0094] Alternatively, the subject method may employ the Cre
recombinase/loxP recognition sites of bacteriophage P1 or the
site-specific FLP recombinase of S. cerevisiae which catalyses
recombination events between about 34 by FLP recognition targets
(FRTs) (Karreman et al. (1996) NAR 24:1616-1624). A similar system
has been developed using the Cre recombinase/loxP recognition sites
of bacteriophage P1 (see PCT/GB00/03837; Vanin et al. (1997) J.
Virol 71:7820-7826).
[0095] In certain embodiments, exposing a cell to a recombinase
comprises turning on the expression of a recombinase encoded by the
cell. The expression of a recombinase may be governed by methods
known in the art. The recombinase may be encoded by a genomically
integrated nucleic acid sequence or from a non-integrated
extrachromosomal expression vector.
[0096] Briefly, in certain embodiments, the recombinase or a
nucleic acid encoding the recombinase may be introduced into the
host cell by transfection, e.g., as described above. Alternatively,
the coding sequence for the recombinase may already be present in
the host cell but not expressed, e.g., because it is under the
control of an inducible promoter. In these embodiments, the
inducible coding sequence may be present on another episomal
nucleic acid, or integrated into the cell's genomic DNA.
Representative inducible promoters of interest that may be
operationally linked to the recombinase coding sequence include,
but are not limited to: aracBAD promoter, the .lamda. pL promoter,
and the like, described above. In these embodiments, the step of
providing the desired recombinase activity in the cell includes
inducing the inducible promoter to cause expression of the desired
recombinase.
[0097] Following production of the desired recombinase activity in
a cell, the resultant cell is then maintained under conditions and
for a period of time sufficient for the recombinase activity to
mediate the inversion of the coding sequence into a 5' to 3'
orientation. In certain cases, the host cell is maintained at a
temperature of between about 20 and 40.degree. C.
[0098] In cases where the subject method exposes the vector to a
unidirectional site-specific recombinase is used, the coding
sequence in the vector inverts into a translatable 5' to 3'
orientation. As noted above, since the first and second
recombination sites are recognizable by a unidirection recombinase
to recombine in only one direction, the inverted coding sequence is
incapable of subsequent recombination.
[0099] In certain cases, the method comprises transfecting into a
cell an episomal circular expression vector comprising a third
recombination site interposed between the first recombination site
and the coding sequence and a fourth recombination site interposed
between the second recombination site and the transcription
termination region, in which the first recombination site and the
second recombination site recombine in the presence of a
recombinase and the third recombination site and the fourth
recombination site recombine in the presence of a recombinase.
[0100] In certain cases, the first and second sites are the first
pair of recombination sites and the third and fourth recombination
sites are the second pair of recombination sites. In these
embodiments, the subject method involves using a vector containing
a first pair and a second pair of recombination sites that are
incompatible with each other to undergo recombination.
[0101] The subject method employing a vector comprising a first
pair and a second pair of recombination sites catalyzes a two-step
recombination process that renders the inverted coding sequence
incapable of subsequent recombination. As explained previously,
this is because after a recombination event, excision of the
intervening sequences between a pair of compatible recombination
sites leaves the coding sequence flanked by incompatible
recombination sites. For example, a first pair of sites may be loxP
sites and the second pair may be lox27722 sites. Certain features
of this recombination process are illustrated by a nonlimiting
example in FIG. 2D and are explained above. In certain embodiments,
the subject method comprises transfecting a vector comprising four
recombination sites as set forth above and exposing the vector to a
recombinase. Recombination ultimately leads to a coding sequence
correctly oriented for production of an expression product and
ensures stable orientation without subsequent excision or perpetual
inversions of the coding sequence.
[0102] The subject method encompasses employing one or more
embodiments of the vector described herein in order to express
genes in a selected group of cells within a population. Since the
expression of the trangene on the vector is dependent on
recombination as described previously, and recombination depends on
the presence of a recombinase, manipulating the availability of the
recombinase in that selected group of cells is a means to control
expression. One embodiment of selective gene expression is to
transfect a vector as described herein in a population containing a
recombinase-expressing cells. This allows only
recombinase-expressing cells to express the transgene.
Alternatively, the method may also encompass selecting a promoter
that is specific for a subset of cells of interest to be used as
the promoter driving the expression of the recombinase or
transgene. In a related embodiment, cell-specific Cre transgenic
mice, for example, may also be used for selective gene
expression.
[0103] Not only can selective gene expression be carried out by
taking advantage of distinct profiles of promoter elements or
recombinase-expressing cell-types in transgenic mice but it can
also be carried out based on cell to cell connection. In a
multicellular organism, there are specific cell types that make
contact with each other. The connection between cells (i.e.
intercellular communication) may be utilized in the subject method
to transfect or deliver the recombinase of interest from one cell
to another.
[0104] For example, the subject method may encompass selectively
targeting specific neurons for expression based on their
topological organization (i.e. input and output targets). Neurons
may be identified based on their projection patterns within the
brain. As another example, the subject method allows the selective
activation of a transgene in a subpopulation of excitatory
pyramidal neurons that project their outputs to the amygdala.
Similarly, other neuronal subpopulations can be effectively
selected for gene expression based on their inputs and/or ouputs.
This can greatly aid in mapping neuronal circuits in a complex
brain structure.
[0105] One way to target selective neuronal population for gene
expression is via the retrograde and anterograde transport
mechanisms used by the neurons. For example, a large number of
neuronal population "A" may be exposed to a vector carrying a
transgene that is not expressed unless in the presence of a
recombinase so that expression of the transgene is considered
recombinase-dependent (e.g. Cre-dependent). Any of the vectors
according to the subject composition may be used. The transgene may
be flanked by one or more recombination sites. The promoter element
driving the expression may also be flanked by one or more
recombination sites. Accordingly, the transgene is expressed only
when the necessary recombinase is delivered to these cells in
population "A". Selective delivery of the recombinase to a
subpopulation of these cells may be carried out via the retrograde
and anterograde transport of an upstream or downstream neuron,
respectively. Details of how this method may be employed using the
retrograde or anterograde transport machinery are set forth
below.
[0106] If one would like to activate the transgene only in a
subpopulation of cells in population "A" that projects to a region
of interest, e.g. neuronal population "B", one would transfect
cells of population "B" (i.e. neurons downstream to "A") with a
recombinase-encoding vector. Particularly, the recombinase is
engineered to be a fusion protein that interacts with the
retrograde transport machinery. Once the neurons in population B
receives such recombinase-encoding vector, the recombinase is
expressed and retrogradely transported upstream only to a selected
group of cells in population "A" that innervate cells in population
"B". As such, retrograde transport may used to selectively
transport the recombinase protein to upstream neurons. Once the
recombinase arrives in those selected cells in population "A", it
may then recombine the vector containing the trangene and allows
transcription and translation of the transgene, in accordance with
the method and composition described previously. In such a manner,
one could control selective gene expression based on the type of
projection destination the cells make.
[0107] For a retrograde-transported recombinase, the recombinase
may be encoded in a viral vector and engineered to fuse to a
retrograde transporting protein such as Rabis virus glycoprotein
(RabiesG). Any other retrograde transporting protein may also be
used.
[0108] On the other hand, in a case where cells in population "B"
projects to cells in population "A", and one is interested
activating a transgene in such a subpopulation of cells in "A" that
receives input from B, one would utilize components of the
anterograde transport for selective gene expression. Population "B"
will be transfected as above (i.e. injected) with the recombinase
fused to an anterograde transporting protein so that the
recombinase travels downstream to neurons in population "A" that
are innervated by "B". As such, only cells in "A" that are
innervated by "B" would receive the recombinase resulting in
subsequent recombination that activates gene expression.
[0109] For an anterograde-transported recombinase, the recombinase
may be encoded in a viral vector and engineered to fuse to a
retrograde transporting protein such as wheat germ agglutinin
(WGA), Phaseolus vulgaris leucoagglutinin (PVL), or Cholera Toxin B
(CTb).
[0110] A more tightly regulated gene expression method may also be
carried out using a combination of any embodiment presented herein.
For example, one or more different recombination sites may be used
on the same or different vectors. E.g. the encoding sequence of a
transgene may be in the reversed orientation as the described for
the subject composition and flanked by one set of a recombination
site while the promoter driving the transgene is also reversed and
flanked by a different set of recombination sites. As such, two
different and incompatible recombinases are required to activate
the trangene. Depending on which group of cells are targeted for
gene expression, each of the different recombinases may be
transported upstream (retrograde) or downstream (anterograde) to
the neuronal population transfected with the recombinase-encoding
vector. Promoters of various strength may also be chosen to
modulate the robustness of the transgene expression desired.
[0111] Using the various embodiments of the subject method, one
could accomplish cell type- and circuit-specific gene expression.
Various strategies for selective gene expression are presented in
FIG. 6. The strategies are formulated for each of the nine
exemplary networks of cells, each containing three populations of
cells (A, B, and C) making different contacts with each other.
Virus that is used in the exemplary networks below refers to virus
carrying a vector encoding a light sensitive channel. In some
networks, there is a cell population that has received light as a
stimulus and is slightly shaded in the figure relative to the other
two populations in the same network. Detailed explanation for
strategies represented in FIG. 6 is set forth below.
[0112] In networks 1 and 2, population B is transfected with a
virus, represented by the hexagon, carrying a vector that encodes a
light sensitive channel (e.g. a microbial opsin). All cells in
population B possess light sensitive channels and the cells they
innervate, two in population C and one in population A are also
affected by active synapses. In network 1, light is shone on
population B, which activate all the light-sensitive cells in
population B. This leads to all of their downstream neurons to be
activated (checkered cells in populations A and C). In network 2,
light is delivered only to population A. The one light-sensitive
cell in population A and its axonal processes are activated
accordingly. As such, the one cell in population B that is
innervated by the stimulated cell in population A is activated.
[0113] In networks 3 and 4, population B is transfected with a
Cre-dependent virus so the expression of the light-sensitive
channel would be dependent on the presence of a Cre recombinase. As
indicated by a triangle present in a cell in population B, Cre is
only expressed in that one cell. As such, only that cell in
population B expresses the light-sensitive channel and is
light-sensitive. The cell it innervates in population C is then
affected by synaptic activity. In network 3, light is delivered to
population B, which causes the light-sensitive cell to release
neurotransmitters. The released neurotransmitter would then lead to
subsequent modulation of the downstream neuron in population C
receiving input. Network 4 is different from network 3 in that
light is delivered to population C as opposed to population B. As
such, light is delivered in the downstream region to which the
light-sensitive neuron is projecting. However, because of how the
cells are connected, the modulation is the same for both networks 3
and 4.
[0114] As for networks 5 and 6, population C is transfected with a
retrograde-transporting virus that carries a vector encoding a
light sensitive channel. Two of the three cells make upstream
connection with two cells in population B. As such, the light
sensitive channel gets retrogradely transported upstream to those
two cells in population B that innervate the two cells in
population C. The two cells in population B then receive the light
sensitive channel and become light-sensitive cells. The one cell in
population C that does not make any connection with any cell in
population B does not affect any cells in population B. Likewise,
cells in population A are not transfected nor do they make any
connection with a cell expressing any transgene so they do not
become light sensitive. Similarly to networks 3 and 4 described
above, although light is delivered to different cell populations in
networks 5 and 6, the modulation outcome is the same.
[0115] In network 7, population C is transfected with a
Cre-dependent virus so the expression of the light sensitive
channel encoded by the viral vector would be dependent on the
presence of the Cre recombinase. Since population B is transfected
with anterograde-transporting Cre, all the cells in population B
expresses Cre and transport Cre to their downstream neurons. Based
on the connections between cells, one of the cells in population B
transports Cre to one cell in population A and two others transport
Cre to two cells in population C. Accordingly, the Cre that gets
transported to two cells in population C would initiate the
expression of the light sensitive channels in two of the cells in
population C.
[0116] As for network 8, population B is transfected with
retrogradely-transporting Cre so all the cells in population B
transports Cre upstream. Two of the three cells transport Cre to
two cells in population A and the third cell to a cell in
population C. Since population A is transfected with Cre-dependent
virus, the two cells that receive retrograde-transported Cre from
population B are able to express the light sensitive channels and
thus become light sensitive. One cell in population A does not have
a retrograde-transported Cre because it is not upstream to any
cells in population B and thus, does not express the
light-sensitive channel. On the other hand, the cells in population
C do not express any light sensitive channels regardless of whether
there is a Cre recombinase or not because they do not possess a
viral vector encoding a light sensitive channel.
[0117] Lastly, in network 9, population A is transfected with
anterograde-transporting Cre and hence, the two cells that are
upstream to cells in population B transport Cre to the cells in
population B. One cell in population A is downstream to the cell in
population B with which it makes a connection so it does not
transport Cre to that cell in population B. Since population C is
transfected with retrograde-transporting Cre-dependent virus, the
two cells in population C transports Cre-dependent virus to the two
upstream cells in population B. Since those two cells in population
B also has Cre that have been transported antegradely from
population A, the two cells in population B express the light
sensitive channels.
[0118] Based on the strategies devised above for various types of
networks, the subject method may be adapted to encompass many other
permutations not listed above. The subject method may be modified
so that a particular cell type or connection may be chosen to
express the transgene of choice.
Kit
[0119] Also provided by the present disclosure are kits for using
the subject composition and for practicing the subject method, as
described above. The subject kit contains a circular expression
vector, comprising i) a promoter; ii) a multiple cloning site for
inserting a coding sequence in a reversed 3'-5' orientation; iii) a
transcription termination sequence; and iv) at least a first
recombination site and a second recombination site flanking the
coding sequence, and instructions for using said vector.
[0120] In certain cases, the kit further comprises cells that are
suitable for transfecting with the subject composition. In certain
cases, the cells are suitable for the propagation of the circular
expression vector. In additional embodiments, the cells contained
in the subject kit may contain a recombinase or a nucleic acid
sequence encoding a recombinase. The recombinase may recognize the
recombination sites in the expression vector and catalyze an
inversion of a intervening sequence between the recombination
sites. The kit may further comprise an inducer to induce the
expression of a recombinase in a cell.
[0121] In certain embodiments, the kit comprises one or more
restriction enzymes. In other cases, the kit further comprises a
map of the enclosed expression vector to aid a user in inserting a
nucleic acid encoding an expression product of interest into a
multiple cloning site.
[0122] In addition to above-mentioned components, the subject kit
typically further includes instructions for using the components of
the kit to practice the subject methods. The instructions for
practicing the subject methods are generally recorded on a suitable
recording medium. For example, the instructions may be printed on a
substrate, such as paper or plastic, etc. As such, the instructions
may be present in the kits as a package insert, in the labeling of
the container of the kit or components thereof (i.e., associated
with the packaging or subpackaging) etc. In other embodiments, the
instructions are present as an electronic storage data file present
on a suitable computer readable storage medium, e.g. CD-ROM,
diskette, etc. In yet other embodiments, the actual instructions
are not present in the kit, but means for obtaining the
instructions from a remote source, e.g. via the internet, are
provided. An example of this embodiment is a kit that includes a
web address where the instructions can be viewed and/or from which
the instructions can be downloaded. As with the instructions, this
means for obtaining the instructions is recorded on a suitable
substrate.
[0123] In addition to the instructions, the kits may also include
one or more control analyte mixtures, e.g., two or more control
analytes for use in testing the kit.
Utility
[0124] The subject invention finds use in a variety of
applications, where such applications generally include, but not
limited to, research applications, polypeptide synthesis
applications, and therapeutic applications.
[0125] Examples of research applications in which the subject
composition finds use include applications designed to characterize
a particular gene with temporal and spatial controls. In such
applications, the vector is employed to insert a gene of interest
into a target cell, the coding sequence is inverted into a correct
translatable orientation when desired, and the resultant effect of
the expressed inserted gene on the cell's phenotype is observed.
The ability to turn on gene expression when desired confers
temporal and spatial control over experimental variables. In this
manner, information about the gene's activity and the nature of the
product encoded thereby can be deduced.
[0126] The subject composition may also be employed to identify and
define DNA sequences that control gene expression, e.g. in a
temporal (e.g. certain developmental stage) or spatial (e.g.
particular cell or tissue type) manner. Yet another research
application in which the subject composition finds use is in the
identification and characterization of the results of gene
expression studies. For example, a plurality of distinct vector
targeted cells (or animals produced therefrom) are prepared in
which the gene of interest is inserted into various targeted cells
in an organism where a recombinase is expressed in different
tissues or at different times. As such, the effects of gene
expression on specific tissues or at different times may be
compared. By plurality is meant at least two, where the number
usually ranges from about 2 to 5000, usually from about 2 to 200.
This plurality of vector targeted cells may be produced by
introducing the vector in a plurality of cells or taking a
collection of pretargeted cells that are homogenous with respect to
the insertion site of the gene, i.e. progeny of a single targeted
cell, and then introducing transposase into one or more of, but not
all of, the constituent members of the collection.
[0127] The subject composition may also be used to study
integration mutants, where a gene of interest is inserted randomly
into the genome and the affects of this random insertion of the
targeted cell phenotype are observed. One can also employ the
subject vectors to produce models in which overexpression and/or
misexpression of a gene of interest is produced in a cell and the
effects of this mutant expression pattern are observed. One can
also use the subject vectors to readily clone genes introduced into
a host cell via insertional mutagenesis that yields phenotypes
and/or expression patterns of interest. In such applications, the
subject vectors are employed to generate insertional mutants
through random integration of DNA. The phenotype and/or expression
pattern of the resultant mutant is then assayed using any
convenient protocol. The temporal and spatial control and the lack
of leakiness may also allow transgenic animals and cells to survive
prior to inverting the gene of interest into a translatable
orientation if such gene expression turns out to be lethal.
[0128] In addition to the above research applications, the subject
composition also finds use in the synthesis of polypeptides, e.g.
proteins of interest. In such applications, a vector that includes
a gene encoding the polypeptide of interest in combination with
requisite and/or desired expression regulatory sequences, e.g.
promoters, etc., (i.e. an expression module) is introduced into the
target cell that is to serve as an expression host for expression
of the polypeptide. Following introduction and subsequent stable
integration into the target cell genome, the targeted host cell is
then maintained under conditions sufficient for expression of the
integrated gene. Once the transformed host expressing the protein
is prepared, the protein is then purified to produce the desired
protein comprising composition. Any convenient protein purification
procedures may be employed, where suitable protein purification
methodologies are described in Guide to Protein Purification,
(Deuthser ed.) (Academic Press, 1990). For example, a lysate may be
prepared from the expression host expressing the protein, and
purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, and the like.
[0129] Useful proteins that may be produced by the subject
invention are, for example, enzymes that can be used for the
production of nutrients and for performing enzymatic reactions in
chemistry, or polypeptides which are useful and valuable as
nutrients or for the treatment of human or animal diseases or for
the prevention thereof, for example hormones, polypeptides with
immunomodulatory activity, anti-viral and/or anti-tumor properties
(e.g., maspin), antibodies, viral antigens, vaccines, clotting
factors, enzyme inhibitors, foodstuffs, and the like. Other useful
polypeptides that may be produced by the methods of the invention
are, for example, those coding for hormones such as secretin,
thymosin, relaxin, luteinizing hormone, parathyroid hormone,
adrenocorticotropin, melanoycte-stimulating hormone,
.beta.-lipotropin, urogastrone or insulin, growth factors, such as
epidermal growth factor, insulin-like growth factor (IGF), e.g.
IGF-I and IGF-II, mast cell growth factor, nerve growth factor,
glial cell line-derived neurotrophic factor (GDNF), or transforming
growth factor (TGF), such as TGF-.alpha. or TGF-.beta. (e.g.
TGF-.beta.1, .beta.2 or .beta.3), growth hormone, such as human or
bovine growth hormones, interleukins, such as interleukin-1 or -2,
human macrophage migration inhibitory factor (MIF), interferons,
such as human .alpha.-interferon, for example interferon-.alpha.A,
.alpha.B, .alpha.D or .alpha.F, .alpha.-interferon,
.gamma.-interferon or a hybrid interferon, for example an
.alpha.A-.alpha.D- or an .alpha.B-.alpha.D-hybrid interferon,
especially the hybrid interferon BDBB, protease inhibitors such as
.alpha..sub.1-antitrypsin, SLPI, .alpha..sub.1-antichymotrypsin, C1
inhibitor, hepatitis virus antigens, such as hepatitis B virus
surface or core antigen or hepatitis A virus antigen, or hepatitis
nonA-nonB (i.e., hepatitis C) virus antigen, plasminogen
activators, such as tissue plasminogen activator or urokinase,
tumor necrosis factors (e.g., TNF-.alpha. or TNF-.beta.),
somatostatin, renin, .beta.-endorphin, immunoglobulins, such as the
light and/or heavy chains of immunoglobulin A, D, E, G, or M or
human-mouse hybrid immunoglobulins, immunoglobulin binding factors,
such as immunoglobulin E binding factor, e.g. sCD23 and the like,
calcitonin, human calcitonin-related peptide, blood clotting
factors, such as factor IX or VIIIc, erythropoietin, eglin, such as
eglin C, desulphatohirudin, such as desulphatohirudin variant HV1,
HV2 or PA, human superoxide dismutase, viral thymidine kinase,
.beta.-lactamase, glucose isomerase, transport proteins such as
human plasma proteins, e.g., serum albumin and transferrin. Fusion
proteins of the above may also be produced by the methods of the
invention.
[0130] Furthermore, the levels of an expressed protein of interest
can be increased by vector amplification (see Bebbington and
Hentschel, "The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in "DNA cloning",
Vol. 3, Academic Press, New York, 1987). When a marker in the
vector system expressing a protein is amplifiable, an increase in
the level of an inhibitor of that marker, when present in the host
cell culture, will increase the number of copies of the marker
gene. Since the amplified region is associated with the
protein-encoding gene, production of the protein of interest will
concomitantly increase (Crouse et al., 1983, Mol. Cell. Biol.,
3:257). An exemplary amplification system includes, but is not
limited to, dihydrofolate reductase (DHFR), which confers
resistance to its inhibitor methotrexate. Other suitable
amplification systems include, but are not limited to, glutamine
synthetase (and its inhibitor methionine sulfoximine), thymidine
synthase (and its inhibitor 5-fluoro uridine),
carbamyl-P-synthetase/aspartate transcarbamylase/dihydro-orotase
(and its inhibitor N-(phosphonacetyl)-L-aspartate), ribonucleoside
reductase (and its inhibitor hydroxyurea), ornithine decarboxylase
(and its inhibitor difluoromethyl ornithine), adenosine deaminase
(and its inhibitor deoxycoformycin), and the like.
[0131] In addition to the utilities described above, the subject
invention may be used to deliver a wide variety of therapeutic
nucleic acids. Therapeutic nucleic acids of interest include genes
that replace defective genes in the target host cell, such as those
responsible for genetic defect based diseased conditions; genes
which have therapeutic utility in the treatment of cancer; and the
like. Specific therapeutic genes for use in the treatment of
genetic defect based disease conditions include genes encoding the
following products: factor VIII, factor IX, .beta.-globin,
low-density protein receptor, adenosine deaminase, purine
nucleoside phosphorylase, sphingomyelinase, glucocerebrosidase,
cystic fibrosis transmembrane regulator, .alpha.-antitrypsin,
CD-18, ornithine transcarbamylase, arginosuccinate synthetase,
phenylalanine hydroxylase, branched-chain, .alpha.-ketoacid
dehydrogenase, fumarylacetoacetate hydrolase, glucose
6-phosphatase, .alpha.-L-fucosidase, .beta.-glucuronidase,
.alpha.-L-iduronidase, galactose 1-phosphate uridyltransferase, and
the like. Cancer therapeutic genes that may be delivered via the
subject vectors include: genes that enhance the antitumor activity
of lymphocytes, genes whose expression product enhances the
immunogenicity of tumor cells, tumor suppressor genes, toxin genes,
suicide genes, multiple-drug resistance genes, antisense sequences,
and the like.
EXAMPLES
[0132] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention nor are they intended to represent that the
experiments below are all or the only experiments performed.
Efforts have been made to ensure accuracy with respect to numbers
used (e.g. amounts, temperature, etc.) but some experimental errors
and deviations should be accounted for. Unless indicated otherwise,
parts are parts by weight, molecular weight is weight average
molecular weight, temperature is in degrees Centigrade, and
pressure is at or near atmospheric.
Methods and Materials
[0133] The following methods and materials were used in the
examples below.
[0134] Plasmids
[0135] The staggered loxP/lox2272 sites were designed using Vector
NTI and synthesized by DNA 2.0 (Menlo Park, Calif.). The gene of
interest (e.g. ChR2-EYFP) was cloned in between the loxP-lox2272
and lox2272-loxP sites using standard molecular cloning techniques.
Briefly, ChR2-EYFP was PCR amplified using primers designed to
append restriction sites to the 5' and 3' ends of ChR2-EYFP.
Subsequently, the ChR2-EYFP PCR product and the construct carrying
the staggered loxP/lox2272 sites were digested using restriction
endonucleases. ChR2-EYFP is then subcloned into the
loxP/lox2272-containing vector backbone via ligation.
[0136] The product containing loxP-lox2272-ChR2-EYFP
(antisense)-loxP-lox2272 was then cloned into a vector containing
the AAV2 ITRs. Also, polyA and WPRE DNA elements were also PCR
amplified from templates and cloned into the AAV vector.
[0137] Virus Production
[0138] Recombinant virus vector were made using specific AAV
serotypes depending on the target tissue (e.g. for gene delivery
into the brain, AAV1, AAV2, or AAV5 were used). Briefly, the
procedure is as follows.
[0139] To transfect one T-225 flask of 293T cells with vectors at a
concentration of 1 .mu.g/.mu.l, the following protocol was used.
For each flask, 63 .mu.l AAV vector, 126 .mu.l pDP1, pDP2, or pDP5
(depending on the desired serotype, for mosaic AAV, use 50:50 of
each serotype plasmid), 510 .mu.l 2M CaCl.sub.2, and 1.45 mL
distilled water were combined to create a DNA mixture. Next, the
DNA mixture was combined with 2.15 mL of 2.times. HEPES buffered
saline (50 mM HEPES, 1.5 mM Na.sub.2HPO.sub.4, 180 mM NaCl, pH
7.05) to create a transfection mixture. Lastly, the transfection
mixture was added to 40.7 mL of Dulbecco's Modified Eagle's Medium
(DMEM) with 10% fetal bovine serum (FBS). The cells were
transfected by incubating the cells with the media mixed with the
transfection mixture.
[0140] After fourteen hours post-transfection, media was removed
and the cells washed once with 20 mL DMEM with 10% FBS (D-10).
Flask was replaced with 40 mL of fresh D10. After seventy-two hours
post-transfection, the cells were collected in 8.5 mL of Tris/NaCl
solution (50 mM Tris, 150 mM NaCl) and frozen in dry ice/ethanol
bath.
[0141] When the cells were ready to be used, the cells were thawed
in 37.degree. C. water bath and 500 .mu.L 10% sodium deoxycholate
monohydrate (Sigma Aldrich, D5670-5G, NaDOC) and 2 .mu.L of
Benzonase (Sigma Aldrich, E8263>=250 U/.mu.L) were added.
Incubation continued for 30 minutes at 37.degree. C. 584 mg of NaCl
were added to the cells. Incubation continued for 30 min at
56.degree. C. The cells were frozen and thawed two more times
before being loaded onto the following discontinuous iodixanol
gradient: 60%-3 mL, 40%-3 mL, 25%-4 mL, 17%-7 mL, Virus-9 mL. Virus
was spun in 70 Ti rotor for 90 minutes at 60,000 rpm and removed by
taking 40% layer, diluted, concentrated in Amicon Concentrator with
PBS-MK (1 mM MgCl.sub.2, 2.5 mM KCl, PBS, pH 7.2), and filtered
through 0.45 .mu.m Acrodisc.
[0142] 10 to 20 .mu.l were analyzed on 10% acrylamide gel and stain
with Comassie Blue Dye. Three bands, VP1 to VP3, were seen (data
not shown).
[0143] Virus Delivery into the Brain
[0144] Recombinant AAV vectors were injected into brain areas of
interest using stereotactic guidance. Surgeries were performed
under aseptic conditions. For anaesthesia, ketamine (16 mg
kg.sup.-1 of body weight) and xylazine (5 mg kg.sup.-1 of body
weight) cocktail were injected intraperitoneally. Fur was sheared
from the top of the animal's head and the head was placed in a
stereotactic apparatus (David Kopf Instruments). A midline scalp
incision was made and a 1-mm-diameter craniotomy was drilled. A
glass micropipette was refilled with 3.0 .mu.L of concentrated
lentivirus solution using a programmable pump (PHD 2000, Harvard
Apparatus) and 1 .mu.L of lentivirus solution was injected at each
site at a concentration of 0.1 mul min.sup.-1.
[0145] Electrophysiology
[0146] Patch-clamp recordings in oocytes and neurons were carried
out as previously described (Nagel, G. et al. Proc. Natl. Acad.
Sci. USA 100:13940-13945 (2003), Nagel, G. et al. Science
296:2395-2398 (2002), and Boyden E. S. et al. Nature Neurosci.
8:1263-1268 (2005)). For whole-cell and cell-attached recording in
cultured hippocampal neurons or acute brain slices, three
intracellular solutions containing chloride were prepared: 4 mM
chloride (135 mM K-gluconate, 10 mM HEPES, 4 mM KCl, 4 mM MgATP,
0.3 mM Na.sub.3GTP, titrated to pH 7.2); 10 mM chloride (129 mM
K-gluconate, 10 mM HEPES, 10 mM KCl, 4 mM MgATP, 0.3 mM
Na.sub.3GTP, titrated to pH 7.2); or 25 mM chloride (114 mM
K-gluconate, 10 mM HEPES, 25 mM KCl, 4 mM MgATP, 0.3 mM
Na.sub.3GTP, titrated to pH 7.2). For cultured hippocampal neurons,
Tyrode's solution was employed as the extracellular solution (125
mM NaCl, 2 mM KCl, 3 mM CaCl.sub.2, 1 mM MgCl.sub.2, 30 mM glucose,
and 25 mM HEPES, titrated to pH 7.3). For preparation of acute
brain slices, mice were killed 2 weeks after viral injection. Acute
brain slices (250 mum) were prepared in ice-cold cutting solution
(64 mM NaCl, 25 mM NaHCO.sub.3, 10 mM glucose, 120 mM sucrose, 2.5
mM KCl, 1.25 mM NaH.sub.2PO.sub.4, 0.5 mM CaCl.sub.2, 7 mM
MgCl.sub.2, and equilibrated with 95% O.sub.2/5% CO.sub.2) using a
vibratome (VT1000S, Leica). Slices were incubated in oxygenated
ACSF (124 mM NaCl, 3 mM KCl, 26 mM NaHCO.sub.3, 1.25 mM
NaH.sub.2PO.sub.4, 2.4 mM CaCl.sub.2, 1.3 mM MgCl.sub.2, 10 mM
glucose, and equilibrated with 95%+O.sub.2/5% CO.sub.2) at
32.degree. C. for 30 min to recover.
Example 1
Construction of the Vector
[0147] In cases where strategies for cell-specific protein
expression depended on the use of endogenous cell-specific
promoters, transcriptional activity based on specific endogenous
promoters was low to moderate and was not adequate for certain
applications. In applications that required the protein of interest
to be expressed at high levels, the use of strong and ubiquitous
promoters was desirable. Some examples of strong promoters included
EF-1a, Ubiq, CAG, CMV, PGK, or the pan-neuronal promoters such as
Synpasin I. However, employing a strong promoter in the context of
a conventional floxed-stop construct might lead to transcriptional
leakiness of the coding region. An exemplary of such floxed-stop
construct is schematically illustrated as the Floxed Stop construct
in FIG. 2A.
[0148] One approach to eliminate the transcriptional leakiness was
to design a vector with the coding region positioned in an
antisense orientation, as illustrated in the single-floxed inverse
ORF (SIO) and double-floxed inverse ORF (DIO) of FIG. 2A. The
coding region was not in the correct translational orientation
unless a recombinase was present to invert the coding region.
[0149] After a coding region had been inverted by a recombinase, if
the recognition sites for recombination were still present, such as
the single pair of inverted loxP sites in SIO of FIG. 2A, perpetual
inversion might occur, resulting in unstable expression of the
coding region. However, if two pairs of staggered incompatible
recognition sites were used to flank the antisense coding region,
as illustrated in DIO of FIG. 2A, a two-step recombination process
permanently flipped the coding region into the sense orientation
relative to the promoter. The two-step recombination process with
the DIO construct is schematically illustrated in FIG. 2D.
Example 2
Comparison of Constructs in HEK293 Cells
[0150] To investigate how the various constructs perform in terms
of their leakiness and expression levels, Floxed-Stop, SIO, and DIO
constructs were expressed in HEK293 cells in the absence and
presence of Cre-recombinase. All constructs contained ChR2-EYFP as
the gene of interest so the level of YFP signal was indicative of
the level of expression.
[0151] Fluorescence micrographs of HEK293 cells expressing the
various constructs in the presence or absence of Cre-recombinase
were shown in FIG. 2B. The left panels represented cells expressing
Cre, and hence the inversion was expected to enable translation of
the coding region, resulting in expressing of ChR2-EYFP. The right
panels represented cells without Cre so that no expression of
ChR2-EYFP was expected to occur.
[0152] The top panels of FIG. 2B showed that cells transfected with
the Floxed Stop construct had a very high YFP signal in the
presence of Cre but also a relatively high YFP signal in the
absence of Cre. This suggested that although Floxed Stop constructs
gave very high expression of the gene of interest in the presence
of Cre, the construct was leaky, leading to high background
signals. Cells transfected with the SIO construct, as shown in the
middle panels of FIG. 2B, had relatively low YFP signal whether or
not Cre was expressed. Interestingly, cells transfected with the
DIO construct showed moderately high YFP signal in the presence of
Cre but virtually no YFP signal in the absence of Cre. This result
indicated that DIO constructs eliminated the leaky expression
associated with the Floxed Stop construct, while allowing stable
expression of the coding region.
[0153] The level of detection sensitivity associated with the
various constructs was determined using FACS. Fluorescence
activated cell sorting (FACS) was used to quantify the population
of cells exhibiting different levels of YFP fluorescence,
transfected with one of the three constructs. The normalized
amounts of cells were graphed against raw YFP fluorescence value as
the x-axis, as shown in FIG. 2C. The graph representing the
population of cells expressing Cre from each of the three
constructs tested was overlaid with a graph of a corresponding
population not expressing Cre.
[0154] FACS analysis suggested that the DIO construct enabled
sensitized detection over background relative to the other
constructs tested herein. For example, by comparing the graphs
representing populations with or without Cre, the greatest
difference between the two graphs in the area where the raw
fluorescence value ranges from 10.sup.10 to almost 10.sup.30, was
observed in the bottom panel of FIG. 2C, where cells were
transfected with the DIO constructs. This large differential
between expression levels tightly regulated by Cre confirms that
DIO constructs enable precise expression control of the gene of
interest and facilitates detection over background.
Example 3
Expression of ChR2-EYFP in Neurons
[0155] A DIO construct was made using ChR2-EYFP as the gene of
interest. The microbial light-sensitive proteins Chlamydomonas
reinhardtii Channelrhodopsin-2 (ChR2-EYFP) allows the bidirectional
control to turn the neurons on and off with high temporal precision
and rapid reversibility. ChR2 is a monovalent cation channel that
allows Na.sup.+ ions to enter the cell following an exposure to
.about.470 nm blue light. Because of its fast temporal kinetics, on
the scale of milliseconds, ChR2 allowed reliable trains of high
frequency action potentials in vivo.
[0156] ChR2-EYFP directed ChR2 channel to be expressed with an
enhanced yellow fluorescent protein as a tag. As shown in FIG. 3A,
left panel, Cre-expressing hippocampal neurons transfected with the
DIO construct containing ChR2-EYFP exhibited robust YFP
fluorescence, an indication that the coding region had undergone
inversion to be in the correct translation orientation. The middle
panel of FIG. 3A showed fluorescence of cells expressing
parvalbumin. Parvalbumin (PV) is present in GABAergic interneurons
in the nervous system, predominantly expressed by chandelier and
basket cells in the cortex. The two fluorescence channel monitoring
ChR2-EYFP and PV were overlaid in the right panel, showing certain
cells that expressed both ChR2-EYFP and PV. The percentage of cells
that were either YFP positive or PV positive were calculated and
shown as bar graphs in FIG. 3B. The fact that almost 100% of the
cells were YFP positive indicated a stable inversion of the coding
region of the DIO construct. The prevalence of YFP signal also
suggested that two-step recombination process described in FIG. 2D
was successful in preventing perpetual inversion subsequent to the
coding region being in the correct translational orientation.
[0157] Further characterization of the DIO construct was carried
out by investigating the functional expression of ChR2 in
Cre-expressing neurons. Voltage-clamped neurons were illuminated by
blue light (473 nm) for a constant period, indicated by the bar
above the current trace in FIG. 3C. The exposure to light evoked an
inward photocurrent, indicating that ChR2 channels were opened in
response to the light stimulus. In another functional assay,
neurons expressing ChR2 were illuminated with brief pulses of blue
light, shown as dashes underneath the voltage trace in FIG. 3D. The
whole-cell recording indicated that action potentials were evoked
precisely with the application of the light stimulus. These results
suggest that ChR2 expressed from the DIO construct were functional
proteins that behaved predictably as ChR2 expressed from previously
used constructs.
Example 4
Circuit-Specific Gene Expression Technology
[0158] The brain consists of numerous cell types interconnected and
embedded within a heterogeneous tissue. Each cell type is
characterized by a unique set of electrophysiological and
biochemical characteristics and the assembly of several different
cell types into a single circuit gives rise to computational units
underlying diverse neurological functions ranging from basic motor
control to complex emotional and cognitive functions.
[0159] One way to interrogate the role of a specific cell type in a
neural circuit may employ light-gated microbial opsins. These
opsins can be used as neural activity regulators since they may be
gated by exposure to brief flashes of blue or yellow light. Three
microbial opsins are shown in FIG. 4, panel A. VChR1 and
channelrhodopsin-2 (ChR2) are capable of exciting neurons using
green and blue light respectively and halorhodopsin (NpHR) is
capable of inhibiting neural activity upon exposure to yellow
light. The bottom left of panel A shows two voltage traces of NpHR
(top trace) and ChR2 (bottom trace), demonstrating the ability of
light flashes to mediate bidirectional optical control of neural
activity. In bottom left of panel A, bars or dashes underneath the
trace represent flashes of light for NpHR or ChR2 respectively. In
the bottom right of panel A, yellow and blue light evoked outward
and inward current respectively in a neuron expressing NpHR and
ChR2.
[0160] The use of these microbial opsins such as ChR2 and NpHR
allow the application of many existing genetic techniques to render
specific sets of neurons light-sensitive, therefore allowing the
control of the function of a set of genetically identical neurons
within the heterogeneously populated brain tissue, without
affecting nearby cells. One example of applying the method
described in the present disclosure is set forth below.
[0161] One purpose may encompass the use of genetically-encoded
neural activity regulators to perturb a selected population of
neurons in the heterogeneously populated brain without affecting
adjacent cells. To achieve strong levels of ChR2 and NpHR
expression in specific neuron populations, a Cre-inducible
Adeno-associated virus (referred to herein as DIO-AAV) was
developed based on a system to decouple cell-specificity from
transcriptional-strength. In this system, the DIO-AAV vector
carries a strong ubiquitous promoter and an inactivated open
reading frame (ORF) in accordance with the embodiments described
previously in the present disclosure. When the virus is delivered
into a transgenic animal expressing the Cre recombinase under a
cell specific promoter, the ORF becomes activated in Cre-expressing
cells (FIG. 2, panel D). In addition to utilizing this expression
system, various approaches may be incorporated to restrict gene
expression to the circuit of interest. The following three
approaches provide solutions to target gene expression with cell
type- and circuit-specificity by leveraging unique properties of
viral and plant/microbial proteins to transport across neurons in a
retrograde or anterograde manner. These systems are also generally
applicable to a variety of mammalian animal models not limited to
mice.
[0162] Exemplary Approach I: Combining Retrograde-Transporting
Viruses and DIO-AAV to Enable Gene Expression in Specific Sets of
Neurons Based on Projection Patterns (Top of FIG. 4, Panel B).
[0163] As the different downstream regions may be involved in
different activities, it may be useful to be able to restrict gene
expression not based on the biochemical marker but also based on
their projection destination. In the prefrontal cortex, for
example, excitatory pyramidal neurons can be divided into different
groups based on their projection destination (e.g. Nucleus
accumbens and amygdala) (Gorelova, N. et al. Neuroscience
76:689-706 (1997); Rosenkranz, J. A. et al. J Neurosci 23:
11054-11064 (2003)). Although all excitatory neurons may be
targeted using the excitatory neuron-specific CaMKIIa promoter
(Aravanis, A. M., et al. J Neural Eng 4:S143-156 (2007)), it is may
be useful to target only the pyramidal neurons that are exclusively
projecting to the brain region of interest. With such control
targeting, only the cortical activity that affects the downstream
region of interest is altered during a behavior test where the
activity of excitatory neurons in the prefrontal cortex is
changed.
[0164] In order to achieve selective gene expression on neurons of
a specific projection destination, a Cre-carrying lentivirus
pseudotyped with the Rabies virus glycoprotein (RabiesG) was
developed. The RabiesG-pseudotyped lentivirus is known to endow
retrograde transport properties to the lentivirus so that the virus
can enter a cell through its axon and travel back to the cell's
nucleus to complete transduction (Watson, D. J. et al. Mol Ther
5:528-537 (2002)). Using this system, the RabiesG-pseudotyped Cre
lentivirus may be delivered downstream where the axons termini are
located and DIO-AAV may be delivered to the brain region where the
cell bodies are located. The Cre lentivirus would travel back to
the cell bodies and activate DIO-AAV only in cells that are
projecting to the injection site of the Cre lentivirus. Unlike
DIO-AAV, which depends on the availability of cell-specific Cre
transgenic mice, this combined system can be applied in any
mammalian animal model.
[0165] More detail of retrograde targeting strategy is presented in
FIG. 5 panels B, C, and D. Panel B shows a schematic illustrating a
strategy for targeting hippocampal dentate gyms neurons sending
projections to the contralateral dentate gyms. Retrograde (Cre-TTC)
transsynaptic Cre virus carrying the construct shown in right of
panel C in FIG. 5 is injected into the ipsilateral dentate gyms. A
Cre-dependent virus is injected into the contralateral dentate gyms
of the same animal.
[0166] Upon expression, Cre-TTC are transynaptically transported to
the upstream neurons to activate Cre-dependent gene expression in
the targeted neurons. Fluorescence images at the right of panel D
in FIG. 5 shows activation of Cre-dependent gene expression in the
contralateral dentate gyms via transsynaptic accumulation of Cre,
although no TTC-Cre is injected into contralateral dentate
gyms.
[0167] In addition to the retrograde-transporting
RabiesG-pseudotyped lentiviral vectors, recombinant Herpes Simplex
Virus-1 (HSV-1) vectors can also be used to achieve retrograde gene
expression in neurons projecting to the vector injection site.
[0168] Exemplary Approach 2: Engineer an Anterograde-Transporting
Cre Recombinase to Achieve an Anterograde-Activating Cre-Inducible
Expression Systems (Middle of FIG. 4, Panel B).
[0169] The RabiesG-pseudotyped Cre lentivirus gives us the ability
to control gene expression in upstream projection neurons through
retrograde gene transfer. However, it is also important to be able
to target gene expression to downstream neurons. For example, in
regions such as the prefrontal cortex which receive innervations
from numerous upstream regions (Gigg, J. et al. Hippocampus
4:189-198 (1994); Akirav, I. et al. Neural Plast 2007:30873
(2007)), one may want to be able to modulate cells that are
innervated by the amygdala independently from the cells that are
innervated by the hippocampus. In this way, the prefrontal cortex's
role in processing emotional input from the amygdala may be
distinguished from memory input coming from the hippocampus.
[0170] To achieve anterograde-specific gene expression, the Cre
recombinase is to be engineered with anterograde transport
properties. This can be accomplished by engineering a fusion of Cre
with an anterograde-transporting protein (referred to herein as
aCre) such as the wheat germ agglutinin (WGA) (Fabian, R. H. et al.
Brain Res 344:41-48 (1985)), Phaseolus vulgaris leucoagglutinin
(PVL) (Cucchiaro, J. B. et al. J Electron Microsc Tech 15:352-368
(1990)), and the (CTb) (Dederen, P. J. et al. Histochem J
26:856-862 (1994)). The recombinant versions of these proteins have
been historically used as neural tracers. In this approach, instead
of using recombinant proteins, an AAV vector carrying aCre is
generated. Similar to how the RabiesG-pseudotyped Cre lentivirus is
delivered in exemplary approach 1 presented above, the aCre AAV
vector is stereotactically delivered upstream and the DIO-AAV
vector downstream. The upstream cells would begin to produce aCre
proteins, which would then be transported through the axon to the
target site and secreted to the postsynaptic neuron to activate
DIO-AAV.
[0171] More detail of anterograde targeting strategy is presented
in FIG. 5 panels A, C, and D. Panel A shows a schematic
illustrating a strategy for targeting hippocampal dentate gyms
neurons receiving projections from the contralateral dentate gyms.
Anterograde (WGA-Cre) virus carrying the construct shown in left of
panel C in FIG. 5 is injected into the ipsilateral dentate gyms. A
Cre-dependent virus is injected into the contralateral dentate gyms
of the same animal.
[0172] Upon expression, WGA-CRE are transynaptically transported to
the downstream neurons to activate Cre-dependent gene expression in
the targeted neurons. Fluorescence images at the left of panel D in
FIG. 5 shows activation of Cre-dependent gene expression in the
contralateral dentate gyms via transsynaptic accumulation of Cre,
although no WGA-Cre is injected into contralateral dentate
gyms.
[0173] In certain cases, anterograde transporting properties may be
enhanced by appending an exporting signal such as the N-terminal
signal peptide from Icam to the Cre fusion protein to facilitate
membrane trafficking of the fusion proteins.
[0174] In the approach disclosed herein, the levels of protein
expression may fine tuned to accommodate various cellular and
experimental systems. This can be done through a combination of
promoter choice, codon optimization, and the use of destabilizing
signal peptides. Since transcriptional strength is decoupled from
the transcriptional specificity in the inducible expression system
disclosed herein, one may tune down the Cre expression level
without compromising the expression level of the protein of
interest, such as ChR2, NpHR and a varieties of genetically-encoded
activity sensors and markers. This method and system can also be
generally applied in all animal models without dependence on
transgenic mice.
[0175] Exemplary Approach 3: Engineer a Combinatorial
Anterograde-/Retrograde-Activating System Using Cre and Flp to
Achieve Cell Type- and Circuit-Specific Expression of Neural
Activity Modulators and Sensors (Bottom of FIG. 4, Panel B).
[0176] The cells in a given downstream region receiving input from
the same upstream region can be quite diverse, and are thought to
be involved in different types of behaviors or different stages of
the same behavior. For example, the excitatory neurons from the
prefrontal cortex projects to the basal lateral and central nucleus
of the amygdale. Some prefrontal cortical neurons selectively
project to the inhibitory intercalated cells and others project to
excitatory neurons in the amygdala. During the presentation of a
fear stimulus, the amygdalar inhibitory and excitatory neurons are
recruited by the prefrontal cortex for different phases of the fear
response such as the acquisition, extinction, and relapse of fear
conditioned responses (Herry, C., et al. Nature 454:600-606
(2008)). Therefore, it may be useful to control the gene expression
of activity modulators (e.g. ChR2 and NpHR) and sensors (e.g. GFP
and GCamp2) in specific types of downstream neurons such as the
amygdalar inhibitory neurons that receive input from the prefrontal
cortex and the amygdalar excitatory neurons that project to the
prefrontal cortex. With such control over gene expression, the
relevant subset of neurons may be modulated or monitored during a
behavior experiment to study their specific involvement.
[0177] Cell type- and circuit-specific gene expression may be
accomplished using two incompatible recombinases such as Cre and
Flp. In this vector system (referred to as DIO2-AAV), the DIO-AAV
vector will be reengineered so that the promoter and the ORF are
both in the reverse orientation (FIG. 4, panel B). The promoter
will be flanked with two sets of incompatible FRT sites and the ORF
will be flanked by two sets of incompatible lox sites as in the
original DIO-AAV design. In an experiment where ChR2 is
specifically expressed in the inhibitory neurons of the amygdala
that are innervated by prefrontal cortical cells, an AAV vector
carrying aFlp (Flp engineered to have anterograde transport
properties) is injected to the prefrontal cortex. In the amygdale,
a mixture of AAV carrying Cre under the inhibitory neuron-specific
VGAT promoter and DIO2-AAV carrying the transgene of interest are
injected. Following viral delivery, although all DIO2-AAV will
non-preferentially enter into all cells in the amygdala, only those
cells that are have both Flp and Cre will be able to express the
transgene of interest. Since Hp will be specifically coming from
the prefrontal cortical projections and Cre expression will be
tightly regulated by the VGAT promoter, only those inhibitory
neurons postsynaptic to the prefrontal cortical cells would express
the transgene of interest. This exemplary method and system is also
generally applicable in all mammalian animal models.
[0178] This approach utilizes the inhibitory neuron-specific VGAT
promoter to drive Cre expression. However, in alternate
embodiments, cell-specific Cre expression may also be driven by a
bacterial artificial chromosome (BAC) transgenic construct.
[0179] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of present invention is embodied by the
appended claims.
* * * * *